Lecture 6c

UV-Vis Spectroscopy

Introduction

• Electromagnetic spectrum

• Visible range: l=380-750 nm• Ultraviolet: l=190-380 nm

Low energyHigh energy

Electronic Transitions

• Most molecules absorb electromagnetic radiation in the visible and/or the ultraviolet range

• The absorption of electromagnetic radiation causes electrons to be excited, which results in a promotion of an electron from a bonding (s or p) or non-bonding orbital (n) to an anti-bonding orbital (s* or p*)

• The larger the energy gap is, the higher the frequency and the shorter the wavelength of the radiation required is (h= Planck’s constant)

• Allowed transitions i.e., s-s*, p-p* are usually strong (large e), while forbiddentransitions (low ) e i.e., n-s*, n-p* aremuch weaker compared to these

• Many transition metal compounds are colored because the d-d transitions fall in the visible range (note that the d-orbitals are not shown to keep the diagram simple)

hc

hE

h= 6.626*10-34 J*sc= 3.00*108 m/s

Color Wheel

• When determining a color, one has to know if the process that causes the color is due to emission or due to absorption of electromagnetic radiation

• Example 1: Sodium atoms emit light at l=589 nm resulting in a yellow-orange flame

• Example 2: Indigo absorbs light at l=605 nm which is in the orange range the compound assumes the complementary color (blue-purple)

What determines the Wavelength?

• Most simple alkenes and ketones absorb in the UV-range because the -p p* and the n-p* energy gaps are quite large

• Conjugation causes a bathochromic shift (red shift)• Increased conjugation often also increases the peak size

as well (hyperchromic)

Compound lmax(nm) e(cm-1*mol-1*L) Chromophore1,4-Pentadiene 178 26000 isolated C=C2-Pentanone 180 900 isolated C=Ob-Carotene 480 133000 conjugated C=C3-Pentenone 224 12590 conjugated C=OAcetophenone 246 9800 conjugated C=O

O

O

O

• The p-p* energy gap for the C=C bond is large• The p-p* and the n-p* energy gap in a C=O bond are both

relatively large as well• The combination of these two

groups affords a new orbital set in which n-p* and the p-p* gaps are much smaller compared in the isolated bonds

• If less energy is required to excite the electrons, a shift tohigher wavelengths for the excitation will be observedi.e., l(n-p*) > l(p-p*)

Conjugation

C=C C=OC=C-C=O

p p

p

p

*p*p

*p

*p

n n

UV-Vis Spectrum of TPCP• Tetraphenylcyclopentadienone

• Bottom line: The exact peak location (l) and absolute peak intensity (e) depend to a certain degree on the solvent used in the measurement

Solvent l(nm) eMethanol 500 1120

  331 6460

  258 24500

Dioxane 504 1410

332 7080

260 26000

Cyclohexane 512 1320

335 7100

262 27100300 nm 600 nm

p-p*330 nm

n-p*500 nm

Beer Lambert Law I

• It describes the attenuation of electromagnetic radiation

• The cell dimension (l) is usually 1 cm • The e-value is wavelength dependent a spectrum is a plot

of the e-values as the function of the wavelength• The larger the e-value is, the larger the peak is going to be• The data given in the literature only list the wavelengths and e-values (or its log value) of the peak maxima i.e., 331 (6460)

• The desirable concentration of the sample is determined by the largest and smallest e-values of the peaks in the spectral window to be measured

lcA **

Beer Lambert Law II

• The absorbance readings for the sample have to be in the range from Amin=0.1 and Amax=1 in order to be reliable

• The concentration limitations are due to • Association at higher concentrations (c>10-4 M)• Linear response of the detector in the UV-spectrophotometer

Linear range

Concentration

Abs

orba

nce

0.1

1.0

cmin cmax

Practical Aspects of UV-Vis I

• Cuvette• It cannot absorb in the measurement window

• Plastic cuvettes absorb more or less in the UV-range already• Most test tubes (borosilicates) start to absorb around 340 nm• Quartz cuvettes have a larger optical window but are very expensive (>$100 each)

• It has to be stable towards the solvent and the compound• Most plastic cuvettes are etched or dissolved by low polarity solvents and can only

be used with alcohols or water• Quartz cuvettes are stable when used with most organic solvents

1. Polystyrene2. Polymethacrylate3. Quartz

detectorPolyethylenecuvette

lamp

Practical Aspects of UV-Vis II

• Solvent

• Hydrocarbons and alcohols possess the largest optical windows • Note that “spectrograde” solvents should be used whenever

possible because many non-spectrograde solvents contain additives i.e., 95 % ethanol contains a lot of aromatics that are active in the UV range

Solvent lower limit (l in nm) Absorbance for l=1 cmAcetone 330 335 (0.30), 340 (0.08), 350 (0.003)Acetonitrile 190 200 (0.10), 210 (0.046), 230 (0.009)Chloroform 265 250 (0.40), 260 (0.05), 270 (0.006)Cyclohexane 210 210 (0.70), 220 (0.32), 230 (0.11), 240 (0.04)Dichloromethane 235 230 (1.30), 240 (0.15), 250 (0.02)Ethanol (abs.) 210 210 (0.70), 220 (0.4), 240 (0.1), 260 (0.009)Hexane 210 210 (0.30), 220 (0.1), 230 (0.03), 240 (0.016)Methanol 210 220 (0.22), 230 (0.1), 240 (0.046), 250 (0.02)Water 191  

Practical Aspects of UV-Vis III

• Important Pointers• Since most measurements require a serial dilution, it is imperative

that the entire compound is dissolved when preparing the stock solution

• For the calculation of the new concentration, the student needs to keep in mind that the total volume is important i.e., if 1 mL of the stock solution was used and 9 mL of additional solvent, the concentration is one tenth of the original concentration

• The student has to run a full spectrum, which requires the software to be set to “spectrum” mode and not to “fixed wavelength” mode (see pop down window in the upper left hand corner)

Practical Aspects of UV-Vis IV

• UV-Vis detector is used in HPLC• The chromatogram changes significantly as the

wavelength is changed because compounds display different e-values at a given wavelength

• The different absorbance characteristics can be used to detector specific compounds and other not

• The linear range for quantitation is limited and given by the wavelength chosen for quantitation